Pharmacological Research 156 (2020) 104792

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Pharmacological Research
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Review

Implication of HMGB1 signaling pathways in Amyotrophic lateral sclerosis T

(ALS): From molecular mechanisms to pre-clinical results
Yam Nath Paudela,*, Efthalia Angelopouloub, Christina Piperib,**, Iekhsan Othmana,
Mohd. Farooq Shaikha,*
a
Neuropharmacology Research Laboratory, Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Bandar Sunway, Selangor, Malaysia
b
Department of Biological Chemistry, Medical School, National and Kapodistrian University of Athens, Athens, Greece

ARTICLE INFO ABSTRACT

Keywords: Amyotrophic lateral sclerosis (ALS) is a devastating and rapidly progressing neurodegenerative disorder with no
HMGB1 effective disease-modifying treatment up to date. The underlying molecular mechanisms of ALS are not yet
ALS completely understood. However, the critical role of the innate immune system and neuroinflammation in ALS
RAGE pathogenesis has gained increased attention. High mobility group box 1 (HMGB1) is a typical damage-associated
TLR4
molecular pattern (DAMP) molecule, acting as a pro-inflammatory cytokine mainly through activation of its
Motor neurons
Neuroinflammation
principal receptors, the receptor for advanced glycation end products (RAGE) and toll-like receptor 4 (TLR4)
which are crucial components of the innate immune system. HMGB1 is an endogenous ligand for both RAGE and
TLR4 that mediate its biological effects. Herein, on the ground of pre-clinical findings we unravel the underlying
mechanisms behind the plausible contribution of HMGB1 and its receptors (RAGE and TLR4) in the ALS pa-
thogenesis. Furthermore, we provide an account of the therapeutic outcomes associated with inhibition/blocking
of HMGB1 receptor signalling in preventing motor neuron’s death and delaying disease progression in ALS
experimental models. There is strong evidence that HMGB1, RAGE and TLR4 signaling axes might present po-
tential targets against ALS, opening a novel headway in ALS research that could plausibly bridge the current
treatment gap.

1. Introduction
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegen-
erative disease also known as Lou Gehrig’s disease, characterized by
rapid degeneration of motor neurons particularly in anterior horns of
the spinal cord and brainstem, as well as of large pyramidal neurons
mainly in the primary motor cortex [1,2]. Epidemiological data have
shown that the current prevalence of ALS is about 5 per 100,000 of the
total population worldwide, and the median survival of ALS patients is
estimated around 24 months in Europe. Despite extensive research ef-
forts towards the elucidation of its pathogenesis, no disease-modifying
treatment is available to date [3].
ALS presents a multifaceted pathophysiology reflecting a complex
interplay between genetic and environmental factors. The vast majority
(90 %) of cases are “sporadic” occurring without a known cause or a
family history, whereas 5–10 % of them are familial-type ALS (FALS)

Abbreviations: HMGB1, high mobility group box 1; RAGE, receptor for advanced glycation end products; TLRs, toll-like receptors; ALS, amyotrophic lateral sclerosis;
FALS, familial ALS; SALS, sporadic ALS; LPS, lipopolysaccharide; fr-HMGB1, fully reduced HMGB1; ds-HMGB1, disulfide HMGB1; ox-HMGB1, sulphonyl HMGB1;
SOD1, Cu2+/Zn2+ superoxide dismutase 1; mSOD1, Mutant SOD1; mSOD1G93A, G93A mutant form of SOD1; SOD1WT, wild‐type SOD1; WT, wild-type; DAMP,
damage-associated molecular patterns; ERK, extracellular signal-regulated kinase; STAT3, signal transducer and activator of transcription 3; AGER, Advanced
glycosylation end‐product specific receptor; PRRs, pathogen recognition receptors; MHC, major histocompatibility complex; BBB, blood-brain barrier; CNS, central
nervous system; DCs, dendritic cells; GFAP, glial fibrillary acidic protein; IL, interleukin; IBA1, ionized calcium-binding adapter molecule 1; iNOS, inducible nitric
oxide synthase; NADPH, nicotinamide-adenine dinucleotide phosphate; NF-κB, nuclear factor κ light chain enhancer of activated B cells; TNF-α, tumor necrosis
factor-α; ChAT, anti-choline acetyltransferase; CD, cluster of differentiation; BDNF, brain-derived neurotrophic factor; GDNF, glial cell line-derived neurotrophic
factor; IR, immunoreactivity
⁎
Corresponding authors at: Neuropharmacology Research Laboratory, Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Jalan
Lagoon Selatan, Bandar Sunway, Selangor, Malaysia.
⁎⁎
Corresponding author at: Department of Biological Chemistry, Medical School, National and Kapodistrian University of Athens, 75M. Asias Street, 11527, Athens,
Greece.
E-mail addresses: paudel@monash.edu (Y.N. Paudel), cpiperi@med.uoa.gr (C. Piperi), farooq.shaikh@monash.edu (M.F. Shaikh).

https://doi.org/10.1016/j.phrs.2020.104792
Received 23 July 2019; Received in revised form 14 February 2020; Accepted 1 April 2020
Available online 08 April 2020
1043-6618/ © 2020 Elsevier Ltd. All rights reserved.
Y.N. Paudel, et al.
associated to genetic mutations which are typically autosomal dom-
inantly inherited [4]. The most common genetic causes of FALS involve
mutations in the genes encoding for superoxide dismutase 1 (SOD1) (20
%), TAR DNA-binding protein 43 (TDP-43) (1–5 %), chromosome 9
open reading frame 72 (C9orf72) (40 %) and fused in sarcoma (FUS)
(1–5 %) [5]. These gene mutations disrupt the degradation of ag-
gregated proteins, impair central nervous system (CNS) glial protective
responses, and promote pro-inflammatory-mediated motor neuron in-
jury [6]. Research advances in ALS genetics and molecular pathogen-
esis, indicate that ALS is rather a syndrome and not a single disorder
[7]. Although the exact etiology of ALS is still largely unknown, accu-
mulating evidence suggests that various cellular and systemic pro-
cesses, such as innate immune system, neuroinflammation, systemic
inflammation, oxidative stress, mitochondrial dysfunction, and ex-
citotoxicity contribute to its pathogenesis [1,7,8].
High mobility group box 1 (HMGB1) acts as a pro-inflammatory
cytokine, as well as an initiator and amplifier of neuroinflammation
that exerts its biological activity mainly through the activation of its
principal receptors, the receptor for advanced glycation end products
(RAGE) and toll-like receptor 4 (TLR4) [9]. An increasing body of
evidence suggests that HMGB1 is implicated in several CNS disorders
such as Parkinson’s disease (PD) [10,11], Alzheimer's disease (AD)
[12], Epileptogenesis [13,14], Multiple sclerosis (MS) [15,16] and ALS
[17] mainly by activation of RAGE/TLR4 signalling axes.
RAGE and TLR4 are two main components of the innate immune
system and HMGB1 acts as an endogenous ligand for both of them [18].
Pre-clinical evidence has shown that HMGB1/RAGE and HMGB1/TLR4
signalling axes are upregulated in ALS, resulting in motor neuron injury
and progression of inflammation [19], suggesting that HMGB1 may
play a pivotal pathogenic role in ALS progression. In this review, we
provide an update of pre-clinical studies investigating the role of
HMGB1, RAGE, and TLR4 in mediating neuroinflammation and disease
progression in ALS. Furthermore, we discuss the therapeutic potential
of HMGB1, RAGE, and TLR4 inhibition/blockade against animal models
of ALS pathogenesis along with future research directions.
2. HMGB1 interaction with RAGE and TLR4 receptors
HMGB1 is the most abundant member of the HMGB family which
consists of HMGB1-4 proteins [20]. HMGB1 is a ubiquitous, highly
conserved nuclear protein with a molecular weight of 25-kDa, con-
sisting of 215 amino acid residues. It is organized in two DNA-binding
domains (termed A and B-boxes) and a negatively charged C-terminal
tail that contains a repeating chain of 30 continuous glutamic and as-
partic acids [21].
HMGB1 is a prototypical alarmin and a pleiotropic danger-asso-
ciated molecular pattern (DAMP) molecule, that is passively released by
damaged cells or secreted from activated immune cells into the extra-
cellular space. HMGB1 can be secreted by activated monocytes, mac-
rophages, and microglial cells in response to several stimuli like lipo-
polysaccharide (LPS), tumour necrosis factor-α (TNF-α) or interleukin-
1β (IL-1β) [22–24]. In turn, secreted HMGB1 can amplify the release of
several cytokines from inflammatory cells and stimulate the release of
HMGB1 from the same cells that activate and maintain the in-
flammatory cascade [22,23].
Moreover, HMGB1 has three isoforms with distinct functions [25].
The first one is fully reduced HMGB1 (fr-HMGB1), consisting of a thiol
group in each of the cysteine residues that induces cell migration. The
second is the disulfide HMGB1 (ds-HMGB1) containing a disulfide
bridge between C23 and C45 and a reduced C106 residue, being a TLR4
ligand. The third isoform is sulphonyl HMGB1 (ox-HMGB1), with
terminally oxidized cysteine residues and no pro-inflammatory activity,
which can induce cell migration or cytokine production [21]. Among
the three isoforms, ds-HMGB1 is the only one contributing to in-
flammatory processes [21].
RAGE is a type 1 transmembrane glycoprotein, cell surface and
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Pharmacological Research 156 (2020) 104792
multi-ligand receptor belonging to the immunoglobulin (Ig) super-
family with a molecular weight of 45 kDa [26]. RAGE is composed of
three Ig domains (V-type, C1-type and C2-type), a transmembrane do-
main, and an intracellular cytoplasmic domain [27]. RAGE is mainly
expressed in endothelial cells, vascular smooth muscle cells, neurons
and macrophages/monocytes [28]. RAGE gene is situated on chromo-
some 6 of the major histocompatibility complex (MHC) class III region,
which contains several other genes crucial for the adaptive and innate
immune responses [29].
RAGE has been recognized as a multi-ligand receptor that can bind
to HMGB1, S100, amyloid-β peptide, DNA, RNA, and other molecules
that regulate several physiological processes [30]. RAGE signaling is
upregulated under conditions of chronic and sustained inflammation
and has emerged as a key regulator of the innate immune response..
Notably, the pathogenic role of RAGE in mediating inflammatory
neurodegeneration has been well-documented [31]. To date, studies
have been mainly focused on the HMGB1/RAGE-induced nuclear factor
kappa-light-chain-enhancer of activated B cells (NF-κB) activation and
the associated inflammatory responses [32]. Moreover, RAGE protein
expression has been found upregulated in brain tissues of patients with
inflammatory neurodegenerative conditions and disorders, including
AD [33], PD [34] and ALS [35]. Accumulating evidence suggests that
RAGE activation, driven by increased availability of its ligands, could
also contribute to ALS pathogenesis [31].
The other HMGB1 receptor, TLR4 is the main member of the TLR
family that responds to bacterial LPS, an essential component of the
outer membrane of gram-negative bacteria [36]. Ten functional TLRs
(TLR1-10) have been identified in humans, which can be divided into
two groups; extracellular TLRs that recognize microbial membrane
components (TLR1, 2, 4, 5, 6, and 10), and intracellular TLRs that re-
cognize microbial nucleic acids (TLR 3, 7, 8, and 9) [37]. TLR4, LPS-
binding protein (LBP), cluster differentiation antigen 14 (CD14) and
myeloid differentiation protein (MD-2) mediate the induction of in-
flammatory responses triggered by LPS. In general, LBP binds to LPS
aggregates leading to the formation of an activated (TLR4/MD-2/LPS)
complex that plays a crucial role in initiating the inflammatory cascade
[38]. TLR4-HMGB1 interaction triggers cytokine production. The
TLR4/MD-2 complex is required for HMGB1-mediated cytokine pro-
duction in macrophages, with ds-HMGB1 being mandatory for this in-
teraction [39,40]. The binding of ds-HMGB1 redox isoform to the TLR4
co-receptor MD-2 resembles LPS binding, but it occurs at a different
position [41]. Notably, TLR4, and its co-receptor CD14 have been
considered as key players of the neuroimmune dysregulation associated
with ALS [19]. Mutant SOD1 (mSOD1) has been reported to bind to
CD14 in ALS, which is a co-receptor of TLR4. Interestingly, microglial
activation mediated by mutant SOD1 can be attenuated using TLR4 and
CD14 blocking antibodies [42].
Among the 14 different receptors of HMGB1 identified to date
[30,43], RAGE and TLR4 are the most extensively studied in HMGB1-
mediated pathologies. Extracellular HMGB1 can interact with several
pathogen recognition receptors (PRRs) to promote cell migration and
cytokine production. The ability of HMGB1 to interact with multiple
unrelated receptors in order to induce diverse functions is tightly
regulated by post-translational redox modifications of the three cy-
steine residues at positions 23, 45, and 106 in the box A and B domains
[21]. Although all redox forms of HMGB1 can bind to RAGE, only ds-
HMGB1 isoforms bind with high affinity [44]. In particular, a struc-
tural-functional study reported that a specific region consisting of the
amino acids 150-183 in the C terminal of HMGB1 is responsible for
RAGE binding [45].
3. Potential role of HMGB1 in ALS pathology
Before moving to HMGB1, it is worth briefly discussing the role of
neuroinflammation in the ALS pathogenesis, which has gained in-
creased attention. Neuroinflammation, mainly characterized by
Y.N. Paudel, et al.
reactive astrocytes and microglia, infiltration of peripheral immune
cells and upregulated inflammatory mediators, has been consistently
observed in motor regions of the CNS in both sporadic and FALS cases
[46]. However, microglia exert both neuroprotective as well as neuro-
toxic effects depending on the stage of the disease progression as well as
on cytokine secretion, indicating that deciphering the role of neuroin-
flammation in ALS is complex but rather promising [47].
HMGB1 can initiate a positive feedback loop, which has been shown
to aggravate the neuroinflammatory and neurodegenerative processes
both in humans as well as in experimental ALS cases [19,48]. The pa-
thogenic role of HMGB1 in ALS has been unravelled from a study using
a SOD1G93A transgenic mouse model (SOD1 mutant with a Gly-93-Ala
substitution), a well-established animal model of FALS [48]. Increased
HMGB1 immunoreactivity (IR) was observed mainly in the cell body
and proximal neurites of labelled motor neurons with no differences
however, been reported between controls and pre-symptomatic
SOD1G93A mice. Extracellular release of HMGB1 was associated with
disease progression, stimulated by cellular damage, thus reflecting the
potential of HMGB1 to act as an extracellular inflammatory signal.
Upon ALS progression, there was a reduction in HMGB1 IR by degen-
erating neurons indicating the extracellular release of HMGB1 [48]. In
accordance, another study showed an increased mRNA expression of
HMGB1 in the lumbar spinal cord of human mutant (h) SOD1G93A
transgenic mice during key disease stages. HMGB1 mRNA levels were
elevated (1.7- fold) at the end-stage of disease, when compared with
wild type (WT) mice. At the protein level, HMGB1 expression was in-
creased at the end-stage of disease by 2.5 fold [49]. Regarding the lo-
calization pattern of HMGB1 in these models, HMGB1 exhibited diffuse
nuclear staining and was mainly co-localised with CD11B-labelled mi-
croglia and glial fibrillary acidic protein (GFAP)-positive astrocytes in
WT mice, whereas no co-localisation with anti-choline acetyltransferase
(ChAT)-positive motor neurons was observed. However, in hSOD1G93A
mice, HMGB1 immunolabelling displayed increased intensity in GFAP-
positive astrocytes and CD11b-labelled microglia. Similar to WT mice,
there was no HMGB1 immunolabelling observed in ChAT-positive
motor neurons of hSOD1G93A mice [49], where ChAT, CD11b, and
GFAP act as cellular markers for motor neurons, microglia and astro-
cytes, respectively. These findings support the pathogenic role of
HMGB1 in ALS, suggesting that HMGB1 may be a key player in the pro-
inflammatory processes that exacerbate disease progression in ALS. The
observed nuclear to cytoplasmic translocation of HMGB1 in the reactive
astrocytes and microglia in ALS patients as well as animal models in-
dicates the plausible pathogenic role for HMGB1 in ALS [19,48,49].
It is of no surprise that astrocytes are highly implicated in neuro-
degeneration and disease progression in ALS [50]. They can be toxic to
motor neurons through the release of neurodamaging molecules
[51,52] or may indirectly contribute to motor neuron degeneration
through loss of their homeostatic and neurosupportive functions
[53,54]. HMGB1-mediated signaling results in an astrocytic production
of neurotrophic factors in normal spinal cord astrocytes, namely brain-
derived neurotrophic factor (BDNF) and glial cell line-derived neuro-
trophic factor (GDNF), whereas dysregulation of astrocytic HMGB1
signaling has been implicated in ALS [17]. Particularly, ALS motor cells
exhibited a nuclear to cytoplasmic translocation of HMGB1 with time,
suggesting that HMGB1 could be also gradually released into extra-
cellular milieu during disease progression. On the other hand, non-
transgenic (non-Tg) and SOD1WT motor neurons exhibited only a basal
nucleus-to-cytoplasm shift of HMGB1 [17]. The potential implication of
HMGB1 signaling in motor neuron-astrocyte interaction in ALS was
further investigated using primary spinal cord astrocytes from non-Tg,
SOD1WT and SOD1G93A transgenic mice exposed to ds-HMGB1 at
asymptomatic stage (30 days), the onset of motor deficits (100 days)
and at symptomatic stage (130 days). In this study, non-Tg and SOD1WT
astrocytes responded to the increased BDNF and GDNF expression,
which was not observed in SOD1G93A astrocytes. Interestingly, non-Tg
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Pharmacological Research 156 (2020) 104792
and SOD1WT astrocytes demonstrated upregulation of RAGE and TLR4,
not observed in SOD1G93A astrocytes, indicating that protective sig-
naling pathways amplified by extracellular ds-HMGB1 in astrocytes
may be disrupted in ALS [17].
In addition to the SOD1G93A transgenic mouse model, the mod-
ulation of HMGB1 levels has been also observed in the Wobbler mouse
model of motor neuron degeneration [55]. It is worth noting that,
Wobbler mice recapitulate all the clinical phenotypes of human ALS
patients [56,57] as well as they exhibit ALS-typical symptoms such as
damage of the motor system [58]. Immunostaining for the HMGB1 and
its principal receptor (TLR4) revealed a significantly higher number of
immunopositive cells in diseased Wobbler mice compared to normal
controls [55]. This upregulation of HMGB1 and TLR4 supports the
notion that neurodegeneration and neuroinflammation co-occur in ALS.
It is worth noting that the correlation between HMGB1 and disease
progression cannot be completely ascertained as the upregulation in the
HMGB1 levels might be attributed both to neuronal death as well as to
disease progression. It is highly possible that the degenerating motor
neurons as well as the associated neuroinflammatory process, induces
HMGB1 release from the activated astrocytes and microglia in
hSOD1G93A mice. Hence, HMGB1 signaling may contribute to the pro-
inflammatory reactions exacerbating disease progression in ALS [49].
HMGB1 plausibly contributes to the progressive inflammatory and
neurodegenerative events in response to neurotoxic settings in the
spinal cord of SOD1G93A mice, rather than presenting a primary event
in the motor neuron death. However, in motor neurons undergoing
degeneration, a downregulation of HMGB1 levels might be a con-
sequence of its passive release upon cellular damage, thus acting as an
inflammatory signal [48].
HMGB1 IR in the cervical spinal cord of control patients was mainly
observed in the cytoplasm of motor neurons, whereas in ALS cases,
residual motor neurons demonstrated variable nuclear/cytoplasmic IR.
Resting glial cells showed nuclear expression in control patients,
whereas cytoplasmic HMGB1 staining was substantially increased in
the glial cells of ALS patients [19]. Interestingly, HMGB1 expression
levels were confined in glial cells, including both astrocytes and acti-
vated microglial cells. Quantification of HMGB1-positive glial cells
confirmed the increased cytoplasmic HMGB1 staining in ALS, as com-
pared to control spinal cord specimens [19]. These findings indicate an
altered subcellular localization of HMGB1 in ALS, supporting the notion
that it could be significantly involved in ALS pathogenesis, possibly due
to its pro-inflammatory effects.
3.1. HMGB1 as a potential biomarker of ALS
The notion of HMGB1 as a potential biomarker of ALS has been
strengthened due to the increased extracellular HMGB1 release in
plasma/serum and tissue during ALS pathology.
Findings from human sporadic ALS and FALS patients reported
upregulated levels of HMGB1 autoantibodies in the serum of ALS pa-
tients as compared to age-matched healthy controls. Intriguingly, the
concentration of anti-HMGB1 autoantibodies was 1.4-fold higher in
female ALS patients compared to male. However, no associations were
detected between anti-HMGB1 autoantibodies levels and onset types,
ALS subtypes, either familiar or sporadic [59].
Similarly, another study that used post-mortem tissues obtained
from 12 sporadic ALS patients demonstrated upregulated HMGB1
mRNA expression in ALS-long term patients (slow disease progression
with long-term survival >48 months) compared to control spinal cord
tissues. In accordance, ALS-short term patients (rapid disease progres-
sion with short-term survival <18 months) displayed similar elevated
HMGB1 levels [19]. These findings suggest that HMGB1 auto Abs could
serve as potential clinical biomarkers for ALS, in monitoring disease
progression.
Y.N. Paudel, et al.
4. The role of RAGE in ALS pathogenesis: Mechanistic insights
from experimental and clinical studies
In the nervous system, the precise role of RAGE is not yet fully
elucidated. However, accumulating evidence demonstrates that RAGE
exerts its biological activities mainly via its implication in partially
cross-linked signaling pathways including extracellular signal-regulated
kinase (ERK), Janus kinase 2 (JAK2)-signal transducer and activator of
transcription 3 (STAT3) and NF-κB, [60]. RAGE is implicated in an
array of cellular neuronal processes, such as neuroinflammation, mu-
tant protein aggregation, production of reactive oxygen species (ROS)
and AGE accumulation [61], contributing to neurodegeneration as well
as to the development of neurodegenerative diseases, including ALS.
There is evidence that RAGE-mediated signaling leads to the increased
synthesis and release of pro-inflammatory molecules and elevation of
nicotinamide-adenine dinucleotide phosphate (NADPH)-dependent
ROS production, resulting in the aggravation of neuroinflammation and
oxidative stress, respectively. These mechanisms exacerbate the pa-
thological processes that lead progressively to neuronal death [62].
Various cell types such as microglia, peripheral monocytes/macro-
phages, astrocytes, T and B lymphocytes are supposed to contribute to
ALS pathology. Interestingly, RAGE is expressed in many of these cell
types and its effects have been studied in both pre-clinical and experi-
mental models of ALS [63].
Experimental in vivo evidence has shed light on the contribution of
RAGE in ALS pathogenesis. In particular, RAGE and mRNA expression
of advanced glycation end product-specific receptor (AGER) levels were
found increased (by 2.24 folds) in lumbar spinal cord specimens of
SOD1 transgenic mice in comparison to controls at the end-stage of the
disease. This evidence suggests that RAGE contributes at least in part, to
the chronic as well as the constant neuronal loss observed in this dis-
order [64].
The putative role of RAGE in ALS has been also investigated in vitro.
The Nerve growth factor (NGF) protein has been found to be post-
translationally modified by oxidation and contribute to RAGE signaling-
mediated motor neuron death, when normal motor neurons are co-
cultured with SOD1 G93A astrocytes. in vitro glycation of NGF has been
shown to favour oligomerization and lead to the generation of RAGE
ligand and modified NGF, further inducing motor neuron death and
astrocyte-mediated motor neuron toxicity [65]. When exosomes from
mutant SOD1-transfected NSC-34 cells were incubated with N9 micro-
glia-like cells, there was an increased upregulation of RAGE, TLR4, IL-
10, and TREM as compared to controls [66]. Moreover, S100β (RAGE
ligand) expression was significantly elevated in C6 rat astrocytoma cells
overexpressing mutant SOD1 G93A protein [67].
A clinical study provides a further molecular glimpse into the con-
tribution of the RAGE/AGE axis in ALS. Quantitative analysis showed
increased RAGE expression, mRNA expression of AGER (the gene en-
coding RAGE) and protein expression of RAGE as determined by
Western blot analysis in the spinal cords of ALS patients when com-
pared to controls, suggesting the possible implication of RAGE pathway
in ALS. These findings imply that selective loss of motor neurons may
be at least partially attributed to the regulation of the RAGE/AGE
pathway in ALS [35]. An upregulation of RAGE expression in reactive
glial cells in both grey and white matter of the spinal cord was de-
monstrated in human sporadic ALS specimens. RAGE was expressed in
reactive astrocytes (GFAP positive cells) and cells of the microglial/
macrophage lineage (HLA-DR positive cells) while a higher RAGE glial
IR score was reported in both ALS-short-term and ALS-long-term pa-
tients as compared to controls [19]. These findings provide strong
evidence regarding the possible role of RAGE signalling in the ALS
pathogenesis.
Soluble RAGE (sRAGE), another potential modulator of inflamma-
tion, has been found reduced in the serum of ALS patients. However, no
positive correlation between the serum sRAGE levels and clinical
parameters of the disease was obtained. This study suggests that sRAGE
4
Pharmacological Research 156 (2020) 104792
might accelerate neurodegeneration and could also be a risk factor for
ALS [68]. Elevated expression of S100β has been also detected in the
serum [69,70], as well as in rat motor neurons exposed to cerebrospinal
fluid (CSF) of ALS patients [70]. This finding reflects the possible
contribution of S100β (RAGE ligand) in ALS and suggest that S100β
might be a potential biomarker [71].
However, it is not well understood whether RAGE interactions in
the spinal cord mediate damage by promoting inflammation, oxidative
stress or other molecular mechanisms. It is worth investigating whether
RAGE and its ligands are the cause, or the modifiers of neurodegen-
erative diseases. Nevertheless, the upregulation of RAGE and its ligands
might present both a potential mechanism or a biomarker [35].
5. Implication of TLR4 signaling in ALS pathogenesis
TLRs, similar to RAGE, are considered as PRRs and their signaling
activates transcriptional factors, cytokine synthesis, and secretion, as
well as promotes inflammatory reactions and brings macrophages and
neutrophils at the site of inflammation through gradient formation
[62]. TLR4, a member of the TLR family, is one of the most crucial
receptors of innate immunity and is widely expressed by immune and
nonimmune cells, including CNS-resident immune cells [72,73]. TLR4
signaling axis can activate two main downstream pathways, one
MyD88-dependent and MyD88-independent, leading to the activation
of NF-κB pathway which in turns activates downstream cytokines, such
as NO, TNF-α, and IL-1β [74]. Activated TLR4 is suggested to promote
the secretion of many cytokines ultimately aggravating the neuroin-
flammatory mechanisms that are involved in ALS [75].
Glial TLR4 signalling has been implicated in disease progression and
contributes to end-stage ALS pathology in hSOD1G93A mice.
Expression levels of TLR4, along with the respective mRNAs, were
shown to be upregulated in microglial and astrocytic cells of mutant
SOD1 mice as compared to WT controls. Interestingly, a prolonged
disease course and a better motor performance were demonstrated in
transgenic mice lacking TLR4, implicating TLR4 as a notable player in
motor neuron degeneration [49].
Extracellular mSOD1 has been shown to cause motor neuron injury
and trigger microgliosis in spinal cord cultures [42]. However, it has
been unclear whether extracellular mSOD1 can directly interact with
motor neurons themselves or whether mSOD1‐activated microglia
mainly contribute to the extracellular mSOD1-mediated motor neuron
neurodegeneration. It has been reported that mSOD1‐associated neu-
rotoxic mechanisms are mediated by microglia and the activation of the
TLR/CD14 pathway involves TLR4 and TLR2 [42], indicating a striking
link between mSOD1-mediated microglial activation and neurodegen-
eration. In addition, granulocyte macrophage-colony stimulating factor
(GM-CSF), a pleiotropic cytokine predominantly released by astrocytes
that up-regulates TLR4 and CD14 expression in microglia, exacerbate
TLR signaling in ALS [76].
Upregulated levels of TLR4 and NF-κB has been observed in a
transgenic model of ALS (SOD1G93A). Particularly, increased TLR4
expression and nuclear protein levels of NF-κB was observed in the
brainstem of SOD1G93A rats compared to WT animals [77]. The acti-
vation of the TLR4/NF-κB cascade is a priming signal that induces the
transcription of inflammasome components. It is known that TLR4 in-
itiates an activation of NF-κB, leading to the activation of the tran-
scription of pro-inflammatory cytokines [78]. This finding highlights
the crucial role of neuroinflammation in ALS.
Clinical findings have also shed more light on the plausible con-
tribution of TLR4 in ALS pathogenesis. More specifically, elevated TLR4
mRNA expression levels were observed in the spinal cord of both
sporadic ALS-short-term and ALS-long-term patients, as compared to
controls. Regarding the cellular distribution of TLR4 in ALS, its staining
intensity was elevated in cells with typical glial morphology in both
white and grey matter, whereas moderate TLR4 IR was detected in
control motor neurons. TLR4 expression was further observed in glial
 Y.N. Paudel, et al.

Table 1
Representative studies reporting HMGB1, RAGE and TLR4 targeted therapies in ALS.
S.N. Interventions Model Treatment schedule Observations References

1 Anti-HMGB1 antibody (2G7)
(100 μg/mouse injection, I.P.)
2 Soluble RAGE (natural competitor
of RAGE)(175 μg/day, I.P.)
G93A
SOD1 transgenic mouse • For early treatment-administered weekly. • Pre-onset treatment with anti-HMGB1 antibody improved hind-limb grip [1]
(C57BL/6 J) model of ALS • For therapeutic treatment- administered 70 days postnatal strength but did not extend survival.
and continued weekly throughout the end stage of the treatment with anti-HMGB1 antibody does not extend survival or
G93A
diseases.
• Post-onset
improved motor performance in SOD1 mice.
• Another cohort of animals received anti-HMGB1 antibody • Treatment with anti-HMGB1 antibody reduced TNF-α and complement C5aR1
from 35 days till 133 days postnatal. gene expression in the spinal cord but does not modulate the activation of glial
cell.
SOD1 transgenic mice model of • Soluble RAGE (175 μg/day I.P.) starting at 8 weeks of age • Soluble RAGE-treated mice demonstrated prolonged life span (138.5 ± 3.1 vs. [2]
ALS until end of an experiment. 130.6 ± 3.1 days), delays progression of ALS and improved functional
performance as compared to murine serum albumin (MSA) treated group.
a Soluble RAGE-treated SOD1 mice, higher neuronal counts and lower
• Inastrocytes has been observed.

5
3 TLR4 inhibitor (TAK-242)
4 TLR4 antagonist
(IAXO102 and FP7)
5 TLR4 antagonism
Transgenic hSOD1G93A mice
model of ALS
In vitro model of ALS
Transgenic hSOD1G93A mice
model of ALS
TAK-242 (3 mg/kg, I.P.), three times per week was
administered to hSOD1G93A mice starting at the age of 7 week.
– •
TLR4−/− female C57BL/6 mice were bred with male
hSOD1G93A to yield hSOD1G93A mice lacking TLR4 (hSOD1G93A
× TLR4−/−).
treatment (n = 13) decreases the clinical disease progression in [3]
G93A
• TAK-242
hSOD1 mice compared with vehicle treated (n = 16) group as evident by
behavioral motor performance analysis (ladder score, hind limb score and
percent survival)
• TAK-242 treatment attenuated motor neuron loss in the spinal cord of
hSOD1G93A mice as well as attenuated spinal cord astrogliosis and microglia
activation in hSOD1G93A mice.
LPS (1 μg/mL) induced motor neuron death was countered by TLR4 [4]
antagonist. IAXO102 (10 μM) and FP7 pre-treatment 2 h before LPS exposure
significantly reduce motor neuron death.
• TLR4 lacking mice (hSOD1G93A × TLR4−/−) improves hind-limb grip [5]
strength at 56, 63, 84 and 161 days of age (n = 8–9, *P < 0.05) and extends
survival when compared to hSOD1G93A mice (184 vs 169, n = 11, *P < 0.05)

HMGB1, High mobility group box 1; ALS, amyotrophic lateral sclerosis; RAGE, Receptor for advanced glycation end products; TLR4, Toll-like receptor 4; SOD1, Cu2+/Zn2+ superoxide dismutase 1; LPS,
Lipopolysaccharide; TNF-α, Tumor necrosis factor-α, I.P., Intraperitoneal; C5Ar1, Complement component 5a receptor 1.
Pharmacological Research 156 (2020) 104792
Y.N. Paudel, et al.
cells of ALS specimens, but not in resting glial cells, depicting the ex-
pression pattern of the protein [19]. These findings strikingly suggest
that the activation of TLR4 axis might demonstrate crucial role in
progression of inflammation, resulting in progressive degeneration of
motor neurons in ALS [19].
These experimental and clinical findings suggest that TLR4 plays a
crucial role in the progressive motor neuron degeneration in ALS, po-
tentially reflecting the significant implication of the innate immune
system in sensing neuronal injury and driving disease progression [79].
6. Potential impact of HMGB1, RAGE, and TLR4 inhibition in ALS
therapy
Since there is no disease-modifying treatment available for ALS to date,
current research has been focused on the exploration of novel effective
therapeutic alternatives. Based on the evidence of HMGB1, RAGE and
TLR4 contribution to ALS pathogenesis, their inhibition/modulation re-
presents a possible therapeutic alternative approach (Table 1).
6.1. HMGB1 blockade in ALS experimental models
Recent advances in HMGB1 biology and its release pattern in the
pathophysiology of several diseases have revealed HMGB1 as a pro-
mising treatment target. A growing body of evidence indicates the
putative role of HMGB1 and its receptor signalling (RAGE/TLR4) in ALS
pathogenesis. However; very few experimental studies have in-
vestigated the therapeutic potential of HMGB1 blockade against ALS.
In this context, Lee et al. have demonstrated that the therapeutic
blockade of HMGB1 before and after disease onset by intraperitoneal (I.P.)
administration of anti-HMGB1 antibody (2G7) (100 μg/mouse injection,
I.P.) reduces early motor deficits in SOD1G93A transgenic mice [18].
Pharmacological blockade of HMGB1 was found to ameliorate hind-limb
grip strength but did not extend survival or improved motor performance
as compared to control antibody-treated SOD1G93A mice. Notably, the
transcripts of Itgam, CD68, Aif1 which are markers of microglia and in-
filtrating monocyte/macrophages as well as the mRNA expression levels of
astrocytic marker, GFAP remained unchanged. On the other hand, tran-
scripts of Ly6c which is a predominant marker of early infiltrating
monocytes/macrophages in lumbar spinal cord were found reduced in
anti-HMGB1 antibody-treated SOD1G93A mice, as compared to control
antibody-treated SOD1G93A mice. These results suggest that anti-HMGB1
antibody treatment does not alter microglia and astrocytes.
In addition, treatment with anti-HMGB1 antibody reduced mono-
cyte markers in the tibialis anterior (TA) and gastrocnemius (GN) as
assessed by the unaltered mRNA expression of macrophage markers
(Itgam, CD68 and Aif1), and decreased mRNA expression of monocyte
marker (Ly6c) in anti-HMGB1 antibody treatment group compared to
control mice [18]. TNF-α mRNA expression and C5a receptor 1 (C5ar1)
were significantly downregulated (0.27-fold and 0.22-fold respectively)
in the spinal cord of the anti-HMGB1 Ab treatment group, compared to
control antibody-treated SOD1G93A mice, whereas mRNA expression
of IL-1β, AGER and TLR4 remained unaffected [18].
However, therapeutic ablation of HMGB1 signalling protects against
early motor deficits but does not extend survival time and demonstrates
overall limited efficacy in the SOD1G93A mouse model of ALS. A possible
reason behind the limited efficacy of HMBG1 neutralization in this study
could be attributed to the presence of other endogenous ligands that
trigger TLR2, TLR4 and RAGE, and ultimately contribute to disease pa-
thology. Moreover, earlier findings have also reported the weak brain and
spinal cord penetrating ability of antibodies [80] that could potentially
alter the inhibitory effect of the compound on CNS-derived HMGB1 [18].
Nevertheless, this study provides evidence suggesting that pharmacolo-
gical blockade/inhibition of HMGB1 might represent a promising ther-
apeutic strategy against ALS. However, the limited number of relative
studies reflects the urgent need of exploring more HMGB1 targeting agents
and evaluating their therapeutic potential against ALS.
6
Pharmacological Research 156 (2020) 104792
6.2. RAGE inhibition in ALS experimental models
Despite the increasing evidence that implicates the contribution of
RAGE in ALS pathogenesis, experimental studies evaluating the ther-
apeutic potential of RAGE inhibition against ALS are still limited.
However, a previous study has reported the significance of RAGE
blockade by sRAGE (175 μg/day, I.P.) in SOD1G93A mutant mice [64].
sRAGE presents a soluble form of RAGE that serves as a natural in-
hibitor, suppressing RAGE function, sequestering RAGE ligands and
blocking their interaction with cell surface RAGE, thus preventing their
subsequent harmful effects on effector cells [81]. sRAGE has been re-
ported to delay disease progression in ALS and was also shown to
prolong the life span of SOD1 transgenic mice, compared to controls
[64]. sRAGE administered to male SOD1G93A mutant mice (starting at
8 weeks until sacrifice) has been shown to extend the life span and
delay the progression of ALS symptomatology [64]. Mice treated with
sRAGE exhibited a higher probability of surviving for a long time after
the onset of the disease and showed a relatively longer lifespan
(138.5 ± 3.1 vs. 130.6 ± 3.1 days), compared to the murine serum
albumin (MSA)-treated group. Moreover, sRAGE-treated mice demon-
strated a higher number of neurons at the end stage of the disease,
compared to the control group. On the contrary, GFAP-im-
munoreactivity was lower in the spinal cord of sRAGE-treated mice as
compared to the MSA-treated ones, reflecting higher neuronal survival
and lower astrocytosis in sRAGE-treated SOD1 mice [64].
Despite the promising effects of RAGE inhibition, the underlying
mechanism of sRAGE-mediated therapeutic effects in ALS remain un-
ravelled and the distinct time courses for initiation of sRAGE adminis-
tration were not tested [63]. Nevertheless, the results of this study
suggest that sRAGE or other forms of RAGE blockade/inhibition might
represent an alternative therapeutic strategy against ALS.
6.3. TLR4 inhibition in ALS experimental models
As discussed above, TLRs (TLR4, TLR2 and their co-receptor CD14)
and especially TLR4 may be highly implicated in ALS pathogenesis. For
instance, TLR4 signaling in pro-inflammatory microglial cells has been
implicated in the degeneration of motor neurons, leading to ALS pa-
thology [82]. Therefore, targeting TLRs has received increasing atten-
tion as a potential novel treatment strategy against ALS.
TAK-242 (3 mg/kg, I.P.), a specific TLR4 inhibitor has been reported
to delay disease progression, attenuate motor neuron loss and decrease
astrogliosis and microglial activation in the spinal cord of hSOD1G93A
mice. These effects were accompanied by attenuation of disease pro-
gression as well as motor improvement, when assessed by motor be-
havioral and ladder testing in TAK-242-treated hSOD1G93A mice. TAK-
242-treated hSOD1G93A mice demonstrated the delayed deterioration
of hind-limb reflex, as compared to vehicle-treated hSOD1G93A mice.
TAK-242 also exerted immunomodulatory effects as shown by the re-
duced serum IL-1β levels in TAK-242-treated hSOD1G93A mice [83].
TAK-242 treatment attenuated motor neuron loss and inhibited spinal
cord astrogliosis and microglia activation in the spinal cord of
hSOD1G93A mice, as evidenced by a significant attenuation of GFAP
and ionized calcium-binding adapter molecule 1 (IBA1), respectively.
Although TAK-242 treatment downregulated mRNA expression of TNF-
α, IL-1β mRNA levels remained unaffected [83].
On a limiting aspect, TAK-242 treatment was found to exert only
moderate and temporary clinical delay in the disease progression
without extending survival in treated animals. Despite of the promising
findings, the study did not evaluate the peripheral effects of TAK-242
treatment on the innervation or neuromuscular junction (NMJ) as
peripheral denervation generally occurs prior to motor neuron death
and symptoms onset in ALS [84]. Hence, this process may have started
before or around the initiation of TAK-242 therapy, thus possibly hin-
dering the beneficial impact of the treatment [83].Nevertheless, these
results suggest that TLR4 manipulation could offer disease-modifying
Y.N. Paudel, et al. Pharmacological Research 156 (2020) 104792

HMGB1, High mobility group box 1; ALS, amyotrophic lateral sclerosis; RAGE, Receptor for advanced glycation end products; TLR4, Toll-like receptor 4; SOD1, Cu2+/Zn2+ superoxide dismutase 1; LPS,
effects against ALS, inferring that targeting of the innate immune

References
system via TLR4 regulation could serve as an emerging therapeutic
strategy against ALS.

[1]

[2]

[3]

[4]

[2]

[1]

[3]
Interestingly, TLR4 deletion/absence provides an extended survival in
the hSOD1G93A mouse models of ALS, further confirming the fact that

lumbar spinal cord tissue from SOD1 transgenic mice reported elevated mRNA expression AGER (2.2-fold higher) compared to normal control.
ALS spinal cord tissue exhibited significantly higher protein levels of HMGB1 compared to control samples (n = 3, *P < 0.05), as determined by western

elevation of HMGB1 mRNA expression was observed in ALS patients with patients with slow disease progression and long-term survival >48

TLR4 mRNA expression was noted in both ALS patients with rapid disease progression and short-term survival <18 months (ALS-ST) and in ALS-
analysis reported elevated expression of RAGE in SOD1 transgenic mice compared to normal control during the end stage of the
upregulation (1.7-fold) in HMGB1 mRNA levels were observed at the end-stage of disease as compared with WT mice (n = 9, **P < 0.01).
TLR4 signalling might contribute to end-stage ALS pathology in
hSOD1G93A mice. hSOD1G93A × TLR4−/− mice demonstrated significant

mice was observed at onset (1.4-folds), mid-symptomatic (1.6-fold) and end-stage (5.6-fold),
extended survival as well as improvement in hind-limb grip strength, in

mRNA expression of AGER (gene encoding RAGE) was reported in the thoracic spinal cords of ALS subjects compared to controls.
comparison to hSOD1G93A mice (184 days vs 169 days, n = 11, *P <

LT patients (patients with slow disease progression and long-term survival > 48 months) as compared to control spinal cord (P < 0.05).
0.05). Nevertheless, survival extension and motor improvement upon TLR4
deletion is only moderate, suggesting that TLR4 is probably only one of

protein expression was upregulated (2.9-fold) at the end-stage of disease as compared with WT mice (n = 4, *P < 0.05).
several contributing factors in hSOD1G93A ALS pathogenesis [49].
In spinal cord primary cultures from SOD1 G93A mice (an in vitro ALS

protein expression was increased (2.5-fold) at the end-stage of disease compared to WT mice (n = 4, *P < 0.05).
model) (as well as in LPS-driven neurotoxicity model, small molecules
IAXO102 (10 μM), FP7 (1 and 10 μM) and extra-virgin olive oil (EVOO) (1
and 10 μg/mL) (TLR4 antagonist) have been assessed for their therapeutic
potential [82]. LPS (1 μg/mL)-induced motor neuron death was counter-
acted by TLR4 antagonists, as evidenced by reduced motor neuron death
upon EVOO treatment. Further pre-treatment of the co-cultures with

immunostaining was upregulated in ALS thoracic spinal cord as compared to control tissue.

increased expression of RAGE in human ALS spinal cord compared to control samples.
IAXO102. FP7 and EVOO significantly prevented the LPS-mediated IL-1β
release, whereas no significant effects were observed for all three TLR4
antagonists on TNF-α release. Pre-treatment with EVOO almost com-
pletely blocked the nitric oxide (NO) release by LPS-stimulated cultures,
whereas IAXO102 and FP7 were not effective. Notably, IAXO102, FP7,
and EVOO reduced the SOD1mut glia-driven motor neuron death [82].

respectively as compared to WT mice (n = 9, *P < 0.05 and ***P < 0.001).

These findings provided compelling evidence that natural TLR4 antago-
nists not only prevent the motor neuron death induced by LPS but also
effectively protect motor neurons from the toxicity of microglia carrying
the SOD1G93A mutation and need to be further confirmed in vivo.

months (LT) as compared to the control spinal cord (P < 0.05).

Similarly, neuroprotective effects of cyanobacteria-derived TLR4 an-
tagonist (VB3323) have been assessed in cultures of the spinal cord and in
an experimental model of motor neuron degeneration [85]. VB3323

G93A
(20 μg/mL) prevented the activation of microglia induced by LPS (1 μg/
mL) as assessed by Western blot analysis. VB3323 also significantly re-

increase in TLR4 mRNA in hSOD1

duced the LPS-dependent cytokine release in co-cultures of motor neurons
and glia, as demonstrated by the downregulation of TNF-α, IL-1β, and IL-
6, reflecting its anti-inflammatory activity. Furthermore, in a mouse model
of motor neuron degeneration (Wobbler mice), VB3323 (5 mg/kg/day,
I.P.) treatment improved their performance in behavioral scores (paw
abnormality degree and the forelegs grip-strength measurements) as
• Immunohistochemical

compared to the Riluzole-treated mouse group. Glial cell activation and

Representative studies reporting upregulation of HMGB1, RAGE and TLR4 in ALS.

TNF-α expression were also reduced in the cervical spinal cord of Wobbler
mice upon VB3323 treatment [85]. Moreover, TLR4 blocking inhibited
• Significant

• Significant

• Significant
• Significant
• Similarly,
• Increased

microglial activation mediated by mutant SOD1G93A. Furthermore,

Observations

diseases.

• Elevated
• blotting.
• HMGB1
• HMGB1

TNF‐α production in mSOD1G93A‐treated microglia was downregulated

• TLR4

Lipopolysaccharide; TNF-α, Tumor necrosis factor-α, WT, Wild-type.

upon administration of TLR4 and TLR2 blocking antibodies [42]. To sum
up, these experimental findings support the role of TLR4 in ALS patho-
genesis and suggest that TLR4 antagonists/inhibitors may be potential
mice model of

Transgenic hSOD1G93A mice model of

therapeutic candidates against motor neuron degeneration in ALS.

SOD1 transgenic mice model of ALS

7. Discussion and future perspectives

ALS is a sporadic multifactorial motor neuron disorder, caused by a

G93A

Human ALS subjects

Human ALS subjects

Human ALS subjects

Human ALS subjects

largely unknown interplay between environmental, genetic and epige-

Transgenic hSOD1

netic modifications [86]. However, the underlying mechanism of motor

neuron death in ALS is still elusive. Among several contributing factors,
Study type

the role of the innate immune system and neuroinflammation are the
most extensively studied [1,6,8].
ALS

ALS

Till date, Riluzole (anti-glutamatergic drug) is the only available

treatment option against ALS that extends survival for approximately 3
Increased levels

months [87]. However, pre-clinical studies investigating the role of

Riluzole in ALS present contradictory results with both beneficial ef-
HMGB1

HMGB1

HMGB1

RAGE

RAGE

TLR4

TLR4

fects in SOD1 transgenic mice models [88], or no effects in disease

progression [89]. These contradictory findings and the lack of effective
Table 2

treatment options against ALS portray the complexity of the disease and
S.N.

reflects the urgent need of developing alternative therapeutic strategies

2

7
Y.N. Paudel, et al.
that not only ameliorate motor symptoms but also improve survival.
Emerging findings demonstrate an increased expression of HMGB1,
RAGE, and TLR4 in reactive glial cells in experimental and human ALS,
implicating the contribution of HMGB1 and its receptor signalling
(RAGE and TLR4) in the pathogenesis of the disease (Table 2).The
distribution and cellular localization of HMGB1and its receptors (RAGE
and TLR4) in the spinal cord of a transgenic mouse model of ALS
strengthen the possibilities of an activation of HMGB1 signaling path-
ways in ALS. HMGB1 signaling cascades leads to aggravation of neu-
roinflammation leading to enhanced synthesis and release of pro-in-
flammatory molecules worsening the existing pathological processes
and resulting in the motor neuron injury in relation to ALS (Fig. 1).
More importantly, upregulation of HMGB1, and its receptors does not
directly cause ALS, but they serve as a risk factors for ALS. The precise
understanding on the plausible link between HMGB1/RAGE/TLR4
signalling in ALS, aggravation of inflammatory cascade and progressive
motor neuron degeneration might represent a novel therapeutic
strategy against ALS mainly by inhibiting neuroinflammation. This is
further supported by experimental studies evaluating the therapeutic
role of HMGB1, RAGE, and TLR4 inhibition/deletion in transgenic ALS
mice. HMGB1, RAGE and TLR4 inhibition/blockade significantly pre-
vented motor neuron death, delayed disease progression, reduced as-
trogliosis and inhibited microglial activation, suggesting that targeting
of HMGB1 and its receptors (RAGE and TLR4) might overcome the
current therapeutic gap, with limited however, supporting findings..
The SOD1G93A transgenic mouse strain offers early onset monitoring
and rapid progression of the disease, presenting with gradual loss of motor
neurons and low variability of the phenotype on defined genetic back-
grounds. In this regard, SOD1G93A transgenic strain represents the most
used animal model for deciphering the early events in the neurodegen-
erative process and understanding the pathogenesis of the disease, as well
as for testing therapeutics designed to halt the progression of ALS [90].
However, despite the knowledge that SOD gene mutation is associated
with FALS, the exact way that mutant SOD1 specifically alters motor
neuron physiology is not completely understood [91]. ALS is an auto-
somal-dominant disorder and all the findings discussed herein are solely
based on the SOD1G93A transgenic mouse model of ALS. Hence, it would
be interesting to investigate the therapeutic outcome of HMGB1, RAGE
and TLR4 inhibition/blockade on other animal models of ALS [92].
In spite of the well-reported beneficial effects of HMGB1 neu-
tralization strategy in a wide range of HMGB1-mediated pathologies
such as Epilepsy [93], MS [94], Traumatic brain injury (TBI) [95] and
Cognitive decline [96], there is only one study till date that has ex-
plored the therapeutic role of anti-HMGB1 antibodies in ALS [18]. This
8
Pharmacological Research 156 (2020) 104792
further reflects the need for more experimental studies evaluating the
therapeutic potential of HMGB1 neutralizing agents, such as anti-
HMGB1 antibodies, natural HMGB1 inhibitors (glycyrrhizin) [97], re-
combinant BoxA, Ethyl pyruvate and Salicylic acid against ALS. Of re-
levance, a potential future HMGB1-based therapeutic strategy could be
the investigation of ds-HMGB1, a specific isoform of HMGB1 that is
considered to primarily contribute to the inflammatory process [98].
Similarly, different available RAGE inhibitors such as sRAGE and ex-
tracellular RAGE inhibitors such as TTP488 and derivatives, FPS-ZM1
[27] should be also evaluated therapeutically against ALS.
Despite the promising outcomes upon therapeutic blockade of
HMGB1 by anti-HMGB1 antibody in ALS, there is also evidence that
anti-HMGB1 antibody treatment provides no beneficial effect on the
motor decline, the survival, the alteration of spinal cord glial numbers
or their activation profiles in SOD1G93A mice [18]. These reflect the
further need of exploring the role of HMGB1 on neuroinflammation and
disease progression in ALS. An abnormal immune system response so-
lely cannot be the causative factor leading to motor neuron death, and
additional factors must be required for disease initiation [1].
Fig. 1. HMGB1 signaling axis in ALS.
HMGB1 has been implicated in the disease progression in ALS
mainly via the interaction with its receptor RAGE and TLR4.
Increased levels of HMGB1, RAGE, AGER (RAGE gene), S100B
(RAGE ligands) and TLR4 have been detected in ALS, sug-
gesting that HMGB1 signaling plausibly contributes to ALS
pathology mainly via aggravating neuroinflammation leading
to motor neuron death. Inhibition/blockade of HMGB1 sig-
naling axis has been shown to delay disease progression, ex-
tend survival, attenuate motor neuron loss and reduce
monocyte markers along with early motor deficits. These
promising findings indicate that HMGB1 signaling axis de-
serves further exploration against ALS that could aid in de-
veloping novel therapeutic strategies.
ALS, Amyotrophic lateral sclerosis; HMGB1, High mobility group
box 1; RAGE, Receptor for advanced glycation end products;
TLRs, Toll-like receptors; AGER, Advanced glycosylation end‐-
product specific receptor; TNF, Tumor necrosis factor; C5ar1, C5a
receptor 1
Beside the pro-inflammatory roles of HMGB1, not much is known
about the initiation mechanism of HMGB1 translocation and release
during disease condition. However, mobilization and release of extra-
cellular HMGB1 occurs not only under pathological conditions but also
under several physiological situations. This reflects the need of better
understanding of the HMGB1 release mechanisms that would eventually
shed lights on the various physiological roles of HMGB1 [99]. Design and
validation of antagonists with greater HMGB1 specificity are crucial along
with the development of antagonists targeting regions of HMGB1 inter-
action with its receptors, representing a future area of research. Hence,
HMGB1-based therapies with negligible off-target effects should be used to
strengthen its value as a specific and effective therapeutic target [100].
In conclusion, the current review sheds light on HMGB1 receptors
signalling in the ALS pathogenesis and reveals the diagnostic and
prognostic biomarker potential of HMGB1, RAGE and TLR4 in ALS,
indicating a new frontier of research.
Author’s contribution
YNP conceived, carried out the literature review and drafted the
manuscript. EA and CP provided critical inputs, revised and edited the
final version of the manuscript. MFS and IO revised the draft. All au-
thors read and approved the final manuscript.
Y.N. Paudel, et al.
Funding sources
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
Ethics approval and consent to participate
Not applicable.
Declaration of Competing Interest
The authors declare that there is no conflict of interest regarding
this work.
Acknowledgement
YNP would like to acknowledge Monash University Malaysia for
supporting with Graduate Research Merit Scholarship.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.phrs.2020.104792.
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